Plasmid-based CTX phage replication system and vibrio cholerae strain that can be infected by CTX phage and can be used for cholera toxin production

ABSTRACT

The present invention relates to a plasmid-based CTX phage replication system and  Vibrio cholerae  strain that can be infected by CTX phage and can be used for cholera toxin production. More particularly, the present invention provides a  Vibrio cholera  variant strain, which expresses a toxT protein in which tyrosine at position 139 is substituted by phenylalanine through the point mutation of a toxT gene using a plasmid-based CTX phage replication system, and is used as a receptor strain which can improve CTX phage infection efficiency and allows a plurality of CTX prophages to simultaneously infect the strain and to be inserted into the chromosome thereof, which the consequent provision of the effect of increasing the production yield of a cholera toxin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of International Application No.PCT/KR2017/014782 filed Dec. 15, 2017, which claims benefit of priorityto Korean Patent Application No. 10-2016-0172681 filed Dec. 16, 2016,Korean Patent Application No. 10-2016-0172684 filed Dec. 16, 2016, andKorean Patent Application No. 10-2017-0144851 filed Nov. 1, 2017, theentire content of which is incorporated herein by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (sequencelisting.txt;Size: 12,789 bytes; and Date of Creation: Jun. 14, 2019) is hereinincorporated by reference in its entirety.

BACKGROUND 1. Field of the Invention

The present invention relates to a plasmid-based CTX phage replicationsystem and Vibrio cholerae strain that can be infected by CTX phage andcan be used for cholera toxin production.

2. Discussion of Related Art

Toxigenic Vibrio cholerae (V. cholerae) is generated by inserting alysogenized CTX phage having a toxin gene into the chromosome of V.cholerae. Serotype O1 and O139 strains, which can be classified intothree biotypes such as classical, El Tor and atypical El Tor, producetoxins, and thus epidemic cholera is developed. Classical biotypestrains have the CTX-cla phage, prototype El Tor strains have CTX-1, andatypical El Tor strains are Wave 2 strains harboring CTX-2 and Wave 3strains harboring CTX-3 to CTX-6. The O139 serotype strains have CTX-1or CTX-O139. Toxigenic strains are generated through infection by a CTXphage, which is a virus having the cholera toxin gene, and subsequentlysogenization thereof. While a model for an evolutionary mechanismthrough which a strain producing a toxin is generated by infection byeach biotype strain has been suggested, the infection and replication ofthe CTX phage are not limited by biotypes of a host strain. Recently,atypical El Tor strains having CTX phages appearing to be formed bymosaics of CTX-cla and CTX-1 have become mainstream worldwide.

The evidence that replication of CTX-1 and CTX-O139 phages is possibleunder laboratory conditions and the evidence of mechanisms forgenerating Wave 2, 3 atypical El Tor strains suggest that replication ofa CTX-cla phage and a similar phage CTX-2 occurs in nature, but is notproved experimentally.

Meanwhile, conventional CTX phage replication was performed using El Torbiotype V. cholerae, in which the CTX phage had been previouslylysogenized (Brigid M Davis et al. Current Opinion in Microbiology,2003, 6: 35-42), and it has been known that CTX^(E1 Tor) (CTX-1) can bemade into a replicative form in a prophage state and can also betransduced. However, a CTX-CTX repeat or CTX-RS1 array is needed, and atthis time, the replicated CTX-1 has been known to be only transduced ina classical biotype strain. The replicative form of CTX-1 has aplasmid-like form (pCTX-1), and the replicative form of CTX-1 transducedinto a classical biotype strain may replicate a CTX phage and transducea different classical strain with the CTX phage.

On the other hand, it has been known that CTX^(cla) has no CTX-CTXarray, and does not replicate because of no transducible E1 Tor strain,and replication of CTX^(cla) and CTX-2 under laboratory conditions hasnot been proven.

SUMMARY OF THE INVENTION

The present invention is directed to providing a recombinant plasmid forreplicating a CTX phage which includes the genomic sequence of a Vibriocholera CTX phage and is replicable in host cells.

The present invention is also directed to providing a host celltransformed by the plasmid.

The present invention is also directed to providing a method ofproducing a Vibrio cholerae CTX phage from the host cells transformed bythe plasmid.

The present invention is also directed to providing a V. choleraevariant strain which can be used as a recipient strain for CTX phageinfection.

The present invention is also directed to providing a method ofimproving the infection efficiency of a CTX phage using the V. choleraevariant strain.

The present invention is also directed to providing a method ofimproving the production yield of cholera toxin using a V. choleraevariant strain harboring a recombinant plasmid for replicating a CTXphage or a CTX prophage.

To achieve the above-described objects, the present invention provides arecombinant plasmid for replicating a CTX phage, which includes thegenomic sequence of a CTX phage in which the full-length sequence of actxA gene represented by SEQ ID NO: 1, and the full-length sequence of actxB gene represented by SEQ ID NO: 2, or fragments thereof aresubstituted with the base sequence of a selection marker gene.

The present invention also provides a host cell transformed by therecombinant plasmid for replicating a CTX phage.

The present invention also provides a method of preparing a CTX phage,which includes isolating and purifying a CTX phage from a culture of thehost cell.

The present invention also provides a V. cholerae variant strain, whichexpresses a toxT protein in which tyrosine (Tyr) at amino acid 139 issubstituted with phenylalanine (Phe) through the point mutation of atoxT gene, and the substituted toxT protein includes the amino acidsequence of SEQ ID NO: 7.

The present invention also provides a V. cholerae variant strain, whichcontains the recombinant plasmid for replicating a CTX phage andexpresses the toxT protein in which Tyr at amino acid 139 is substitutedwith Phe through the point mutation of the toxT gene, and thesubstituted toxT protein includes the amino acid sequence of SEQ ID NO:7.

The present invention also provides a V. cholerae variant strain, whichis infected by a V. cholerae strain harboring one or more CTX prophagesselected from the group consisting of CTX-1, CTX-cla, CTX-2, CTX-env andCTX-O139 to insert the genomic sequence of a CTX phage into itschromosome, and expresses a toxT protein in which Tyr at amino acid 139is substituted with Phe through the point mutation of a toxT gene, andthe substituted toxT protein includes the amino acid sequence of SEQ IDNO: 7.

The present invention also provides a method of preparing a V. choleraevariant strain, which includes inducing a UAU to UUU point mutation atthe 139^(th) codon of a toxT gene of a V. cholerae strain to express atoxT protein in which Tyr at amino acid 139 is substituted with Phe.

The present invention also provides a method of improving the infectionefficiency of a CTX phage, which includes transducing the V. choleraevariant strain by the recombinant plasmid for replicating a CTX phage,or performing infection using a V. cholerae strain harboring one or moreCTX prophages selected from the group consisting of CTX-1, CTX-cla,CTX-2, CTX-env and CTX-O139 as a donor strain and the V. choleraevariant strain as a recipient strain.

The present invention also provides a method of improving the productionyield of cholera toxin, which includes single-phase culturing the V.cholerae variant strain under conditions of 30 to 37° C. and pH 6 to 8.

The present invention can improve the CTX phage infection efficiency fora V. cholerae variant strain expressing a toxT protein in which Tyr atamino acid 139 is substituted with Phe through the point mutation of atoxT gene as a recipient strain using a plasmid-based CTX phagereplication system, simultaneously infect a plurality of CTX prophages,and increase the production yield of cholera toxin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the genetic map of a CTX phage and the construct of apUC-CTX plasmid. The recombinant pUC-CTX plasmid consists of three DNAfragments, that is, a replication origin fragment of pUC18, a DNAfragment spanning from nucleotide 245 of zot to the termination codon ofrstR of RS1 or CTX phage at downstream of zot amplified from PM8, and afragment spanning the att sequence of CTX phage or from nucleotide 119nucleotide upstream of the termination codon of rstR to nucleotide 244of zot. Therefore, the zot-3′UTR fragment is common to all constructs,and the 5′UTR-zot fragments are phage type-specific.

FIG. 2 shows the western blotting results comparing the expression ofTcpA serving as a recipient during CTX phage infection in a strainincluding 139Y at toxT and a strain in which a 139F point mutation isintroduced: Lane 1: O395; Lane 2: MG116025; Lane 3:MG116025-toxT^(F139Y); Lane 4: IB4122; Lane 5: IB4122-toxT^(Y139F); andLane 6: A213, Lane 7: A213-toxT^(Y139F).

FIG. 3 shows the result of Coomassie Brilliant blue staining fordetermining the expression of TcpA serving as a recipient during CTXphage infection in in a strain having 139Y at toxT and a strain in whicha 139F point mutation is introduced and the full-size Western blot imageof the TcpA expression, wherein the arrow indicates TcpA.

FIGS. 4A-4C show the electron microscopy images of CTX phages: FIG. 4Ais a CTX-1kan phage produced from O395 transduced by pCTX-1kan; FIG. 4Bis a CTX-2kan phage replicated from a MG116025 transductant bypCTX-2kan; and FIG. 4C is a CTX-O139kan phage produced from an O395transductant by pCTX-O139kan (100,000×).

FIG. 5 shows the result of immunoblot analysis of CtxA production. Lanes1 and 2: O395; Lanes 3 and 4: MG116025; Lanes 5 and 6:MG116025-toxT^(F139Y); Lanes 7 and 8: IB4122; Lanes 9 and 10:IB4122-toxT^(Y139F); Lanes 11 and 12: A213; and Lanes 13 and 14:A213-toxT^(Y139F). The odd-numbered lanes represent samples grown in LBmedia at 37° C., the even-numbered lanes represent samples grown in LBmedia at pH 6.5 and 30° C., and the arrow indicates CtxA.

FIGS. 6A and 6B show the analysis results of CtxA expression: FIG. 6Ashows the Western blot image of CtxA expression; and FIG. 6B shows thegel image of the same samples used in the Western blotting, which arestained by Coomassie Brilliant blue staining. Lanes 1 and 2 are O395,Lanes 3 and 4 are MG116025-toxT-139F, Lanes 5 and 6 areMG116025-toxT-139Y, Lanes 7 and 8 are IB4122-toxT-139Y, Lanes 9 and 10are IB4122-toxT-139F, Lanes 11 and 12 are A213-toxT-139Y, and Lanes 13and 14 are A213-toxT139F. The odd-numbered lanes represent samplescultured in LB media at 37° C., the even-numbered lanes representsamples cultured in LB media (pH 6.5) at 30° C., and the arrow indicatesCtxA.

FIGS. 7A and 7B show the process of producing pCTX-2 by recombinationbetween pCTX-1 and a tandem repeat of a CTX-2 prophage: FIG. 7A isregarding rstR exchange between pCTX-1-kan-N1 and a tandem repeat ofCTX-2 in B33, and FIG. 7B is regarding the potential of generatingpCTX-2 by recombination between pCTX-1 and a tandem repeat of CTX-cla ina hypothetical classical strain.

FIG. 8 shows the CTX array in classical biotype strains O395 and PM37.

FIG. 9 shows the CTX arrays of MG116025 and derivative strains thereof:PM25˜PM29 are constructed by the stepwise removal of CTX-1, RS1 and TLCfrom MG116025, and PM30˜PM36 are constructed by the insertion ofCTX-2-kan-N2 or and CTX-1-kan-N2 in parental strains.

FIG. 10 shows the CTX arrays of strain B33 and derivative strainsthereof: PM38 is constructed by inserting pCTX-1-kan-N2 into chromosome1 of B33, PM21 is constructed by removing a CTX-1 prophage fromchromosome 2 of B33, and PM39 and PM40 are constructed by insertingpCTX-1-kan-N2 and pCTX-2-kan-N2 into chromosome 1 of PM21, respectively.

FIG. 11 shows the CTX arrays constructed from A213, and various strainsconstructed from non-toxic strain A213 containing only TLC todemonstrate the concept of designing and constructing strains by usingvarious CTX arrays.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Generally, CTX^(El Tor) (CTX-1) is able to be generated from a prophagestate to a replicative form and also generated by transduction. However,it has been known that a CTX-CTX repeat or CTX-RS1 array is required,and only classical biotype strains can be transduced by replicative-formCTX-1 phages. In addition, the replication of CTX^(cla) (CTX-cla) hasnot been demonstrated under laboratory conditions because of no CTX:CTXor CTX:RS1 array and no El Tor strain to be transduced.

Therefore, the inventors constructed a plasmid-based CTX phagereplication system for CTX-cla under laboratory conditions and an El Torvariant strain to be transduced by a CTX phage to demonstrate areplication process.

Accordingly, the present invention relates to a recombinant plasmid forreplicating a CTX phage, which includes the genomic sequence of a CTXphage in which the full-length sequence of a ctxA gene of SEQ ID NO: 1and the full-length sequence of a ctxB gene of SEQ ID NO: 2 or fragmentsthereof are substituted with the base sequences of selection markergenes.

In the present invention, a plasmid is constructed as a unit forreplicating a CTX phage, amplified in E. coli, and then injected into V.cholerae to be replicated. The CTX phage generated from the plasmid isthe same as a phage generated in nature. The genome of the CTX phageinjected into a plasmid is capable of being artificially synthesized.Since the CTX replication can occur in a plasmid, both CTX^(El Tor) andCTX^(cla) can be produced. This suggests that different CTX phages(CTX-2, CTX-O139 and CTX-env) may be produced. As the El Tor straincapable of being transduced by CTX^(cla) was newly found, it can beconfirmed that CTX^(cla) can be transduced.

El Tor strains have been previously known to be transduced by CTX-1 in aliving body (specifically, in the intestine of a mouse), but were notdemonstrated to be transduced under laboratory conditions. Therefore, itcan be expected that, only when suitable laboratory conditions areprovided, the El Tor strains may be transduced by CTX-1 as well asCTX-cla or CTX-2. Since CTX-2 having rstR^(cla), as a tandem repeat, wascontained on chromosome 2 of a Wave 2 atypical El Tor strain, CTX-2replication can be expected, but had not been experimentallydemonstrated. It can also be considered that this is because there wasno El Tor strain transduced by CTX-2 or CTX-cla.

The recombinant plasmid for replicating a CTX phage of the presentinvention will be described in detail as follows.

The recombinant plasmid for replicating a CTX phage includes the genomicsequence of a CTX phage in which the full-length sequence of a ctxA geneand the full-length sequence of a ctxB gene or fragments thereof aresubstituted with a selection marker gene.

The genomic sequence of the CTX phage may be one or more genomicsequences of CTX-1, CTX-cla, CTX-2, CTX-env and CTX-O139.

In one exemplary embodiment of the present invention, the genome of theCTX-1 phage consists of rstR, rstA, rstB, cep, orfU, ace, zot, ctxA andctxB genes.

In the recombinant plasmid for replicating a CTX phage of the presentinvention, the full-length sequence of the ctxA gene and the full-lengthsequence of the ctxB gene or fragments thereof may be substituted with aselection marker gene cassette.

The full-length sequence of the ctxA gene may be the base sequences ofSEQ ID NO. 1. In addition, the full-length sequence of the ctxB gene maybe the base sequences of SEQ ID NO: 2. The fragment of the ctxB gene mayinclude the base sequences of SEQ ID NO: 3.

In addition, a selection marker gene such as a drug-resistance genefacilitates the detection of a transductant due to a phenotype of theselection marker gene in the transductant. As the selection marker gene,an ampicillin-resistance gene, a kanamycin-resistance gene, astreptomycin-resistance gene, a tetracycline-resistance gene, anerythromycin-resistance gene or a chloramphenicol acetyl transferasegene may be used. More specifically, in the present invention, thekanamycin-resistance gene may be substituted for the full-lengthsequence of the ctxA gene and the full-length sequence of the ctxB geneor fragments thereof. More specifically, the substitutedkanamycin-resistance gene may include base sequences represented by SEQID NO: 4.

According to one embodiment of the present invention, the part in whichthe full-length sequence of the ctxA gene and the full-length sequenceof the ctxB gene or fragments thereof are substituted with thekanamycin-resistance gene further includes 5′ and 3′-non-codingsequences, and represented by sequences of SEQ ID NO: 5.

The recombinant plasmid for replicating a CTX phage of the presentinvention may contain a region essential for self-replication of theplasmid (a replication regulatory region, or a gene expressioncassette). Even when a region other than the replication regulatorysequence, that is, a region excluding a replication origin and a regionincluding genes necessary for replication, is deleted, the recombinationplasmid may be replicated in host cells.

The replication regulatory region is a nucleic acid sequence including apromoter, and having an expression activity of regulating theexpression, that is, transcription and translation, of a gene afterbeing functionally linked to the gene subjected to expression.

The recombinant plasmid for replicating a CTX phage may be derived froma E. coli-derived plasmid for transformation selected from the groupconsisting of pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398,RSF1010, pMW119, pMW118, pMW219 and pMW218.

The recombinant plasmid for replicating a CTX phage may be constructedin the same manner as known constructs such as conventional cloningvectors, expression vectors, etc.

For the preparation of plasmid DNA, the cleavage and binding of DNA andtransformation, methods known by those skilled in the art may be used.The methods are disclosed in the literature [Sambrook, J., Fritsch, E.F., and Maniatis, T., “Molecular Cloning: A Laboratory Manual, SecondEdition”, Cold Spring Harbor Laboratory Press (1989)].

According to one embodiment of the present invention, the recombinantplasmid for replicating a CTX phage may be prepared by operably linkinga selection marker gene cassette to the genomic sequence of a CTX phagefrom which the full-length sequence of a ctxA gene and the full-lengthsequence of a ctxB gene or fragments thereof are removed, and insertingthe DNA fragment into the replication origin of the plasmid.

More specifically, the recombinant plasmid for replicating a CTX phagemay include:

a replication origin fragment of a plasmid;

a DNA fragment including a sequence spanning nucleotide 245 of zotupstream of the genome of a CTX phage to the termination codon of rstRdownstream thereof, a part of the zot gene, a selection marker genecassette, and a partial sequence up to 3′UTR of the ctxB gene in acholera strain having a CTX:RS1 or CTX:CTX array; and a DNA fragmentincluding 5′UTR of a CTX phage, rstR, rstA, rstB, cep, orfU and asequence up to nucleotide 244 of zot may be included in a cholera strainhaving an array with CTX at the very front. The DNA fragment may beamplified in a different type of CTX phage (CTX-1, CTX-cla, CTX-2,CTX-O139 or CTX-env) and linked to two different fragments such thatdifferent types of CTX phage genomes may be contained, respectively.

An example of the recombinant plasmid for replicating a CTX phage of thepresent invention is illustrated in the cleavage map of FIG. 1.

The recombinant plasmid for replicating a CTX phage of the presentinvention may be replicated in E. coli, the genus Salmonella, the genusShigella, the genus Klebsiella, the genus Pseudomonas or the genusVibrio. More specifically, the recombinant plasmid for replicating a CTXphage of the present invention may be E. coli or the genus Vibrio.

The genus Vibrio may include a classical biotype, an E1 Tor biotype, anatypical E1 Tor, an O139 serotype, or any serotype of V. cholerae. Mostspecifically, CTX phages can be replicated by transducing a classicalbiotype strain by a CTX-1 genome-cloned plasmid. A CTX-cla or CTX-2genome-cloned plasmid can be introduced into any El Tor strain throughtransformation, thereby replicating a CTX-cla or CTX-2 phage, and thereplicated CTX phage can be introduced into a Vibrio cholera strain inwhich a Tyr139Phe mutation is present at a toxT gene by transduction toreplicate the CTX phage. Such variant strains may include anA213-toxT^(Y139F) strain or an IB4122-toxT^(Y139F) strain, but thepresent invention is not limited thereto.

Therefore, the present invention provides a host cell transformed with arecombinant plasmid for replicating a CTX phage.

A method of transforming host cells with the recombinant plasmid forreplicating a CTX phage may be selected from all transformation methodsknown in the art without limitation, for example, selected frombacterial protoplast fusion, electroporation, and infection using aviral vector.

The host cells may be selected from E. coli, the genus Salmonella, thegenus Shigella, the genus Klebsiella, the genus Pseudomonas or the genusVibrio.

The culture of the transformed host cells may be performed in suitablemedia by various methods known in the art. Examples of the culturemethod include batch, continuous and fed-batch cultures. The fed-batchculture may include injection batch and repeated injection batchcultures, but the present invention is not limited thereto.

The medium used herein generally includes one or more of carbon sources,nitrogen sources, inorganic salts, vitamins and/or trace elements. Apreferable carbon source is a saccharide such as a monosaccharide, adisaccharide or a polysaccharide. A nitrogen source is generally anorganic or inorganic nitrogen compound, or a material including acompound thereof. Examples of the nitrogen sources include an ammoniagas, an ammonium salt such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate or ammonium nitride, a nitrate,urea, an amino acid, or a complex nitrogen source, such as a corn steepliquor, soybean powder, a soy protein, a yeast extract or a meatextract. The nitrogen source may be used alone or in combination.Inorganic compounds that can be contained in media include chlorides,phosphates or sulfates of calcium, magnesium, sodium, cobalt,molybdenum, potassium, manganese, zinc, copper and iron. As aphosphorous source, phosphoric acid, potassium dihydrogen phosphate ordipotassium hydrogen phosphate, or a sodium-containing saltcorresponding thereto may be used. To maintain a metal ion in asolution, a chelating agent may be added to the medium. Morespecifically, to enhance the replication of the CTX phage, nanomoles ofmitomycin C may be added. All components in the medium are sterilized byheating (at 1.5 bar and 121° C. for 20 minutes) or sterile filtration.These components may be sterilized together or independently as needed.All components of the medium may be present at the beginning of theculture, or may be arbitrarily added by way of continuous or batchculture.

The CTX phages may be produced by isolating and purifying CTX phagesfrom the transformed cell mass, culture or lysate of the host cells orthe lysate of the culture.

Therefore, the present invention provides a method of producing CTXphages, which includes isolating and purifying CTX phages from a cultureof the host cells.

In addition, the present invention relates to a V. cholerae variantstrain, which expresses a toxT protein in which Tyr at amino acid 139 issubstituted with Phe through the point mutation of a toxT gene, and thesubstituted toxT protein includes the amino acid sequence of SEQ ID NO:7.

The present invention provides a method of preparing a V. choleraevariant strain, which includes inducing a UAU to UUU point mutation atthe 139^(th) codon of toxT gene of a V. cholerae strain to express toxTprotein of SEQ ID NO: 7 in which Tyr is substituted with Phe at aminoacid 139.

According to one embodiment of the present invention, the inventorsfound out a strain transduced by CTX-2 and CTX-cla among El Tor biotypestrains. Compared with other V. cholerae strains, the El Tor strain hasone single nucleotide polymorphism (SNP) at a toxT gene, which is atranscription activator of tcpA and ctxAB. Other El Tor strains have Tyrat amino acid 139 (SEQ ID NO: 6), whereas this strain has Phe at aminoacid 139 of the toxT gene (SEQ ID NO: 7). Due to such a mutation, thestrain may be transduced by a CTX phage and thus produce cholera toxinunder laboratory conditions. When SNP of toxT has changed from 139Phe tothe toxT allele (139Tyr) of a different El Tor strain, the stain of thepresent invention does not act as a transduction recipient strain. WhenSNP (139Phe) is introduced into toxT of other El Tor strains, thesestrains may be infected by CTX phages. Since toxT is directly involvedin transcriptional activation of both tcp expression and ctxABexpression, when El Tor strains were cultured in a single phase, it wasconfirmed that toxin expression was also increased. In addition, using aplurality of recombinant plasmids for replicating a CTX phage, an E1 Torstrain having all of multiple CTX prophages can be constructed.

As a strain used to manufacture the V. cholerae variant strain of thepresent invention, a classical biotype, E1 Tor biotype, atypical E1 Torbiotype or O139 serotype V. cholerae strain may be used.

The V. cholerae variant strain may be any one of V. cholerae strainsexpressing the toxT protein of SEQ ID NO: 7 in which Tyr at amino acid139 is substituted with Phe through the point mutation of a toxT genewithout limitation, and may be, for example, a MG116025 (Matlab typeIII)-toxT^(Y139F) strain, a B33-toxT^(Y139F) strain, anA213-toxT^(Y139F) strain or an IB4122-toxT^(Y139F) strain, but thepresent invention is not limited thereto.

The V. cholerae variant strain of the present invention may include aCTX prophage selected from CTX-1, CTX-cla, CTX-2, CTX-env, CTX-O139 anda combination thereof.

One or more of the CTX prophages may be included by transducing a V.cholerae variant strain by the above-described recombinant plasmid forreplicating a CTX phage selected from the group consisting of CTX-1,CTX-cla, CTX-2, CTX-env and CTX-O139; or infecting a V. cholerae variantstrain using a V. cholerae strain which contains one or more CTXprophages selected from the group consisting of CTX-1, CTX-cla, CTX-2,CTX-env and CTX-O139 as a donor strain.

Accordingly, the present invention provides a V. cholerae variantstrain, which includes the recombinant plasmid for replicating a CTXphage and expresses a toxT protein in which Tyr at amino acid 139 issubstituted with Phe through the point mutation of a toxT gene, whereinthe substituted toxT protein includes the amino acid sequence of SEQ IDNO: 7.

In addition, the present invention provides a V. cholerae variantstrain, which is infected using a V. cholerae strain harboring one ormore CTX prophages selected from the group consisting of CTX-1, CTX-cla,CTX-2, CTX-env and CTX-O139 such that the genomic sequence of the CTXphage is inserted into the chromosome of the strain, and expresses atoxT protein in which Tyr at amino acid 139 is substituted with Phethrough the point mutation of a toxT gene, wherein the substituted toxTprotein includes the amino acid sequence of SEQ ID NO: 7.

The “prophage” used herein may be a non-infectious type, and present ina phage state maintained in V. cholerae cells.

The “replicative-form CTX phage (pCTX)” is not inserted into a specificregion of the V. cholerae chromosome and present in the form of aplasmid outside the chromosome.

The “donor strain or donor” used herein is a donor of a V. cholerae CTXphage, and refers to a V. cholerae strain in which a CTX phage isinserted into the chromosome as a lysogenic phage (lysogen), or a V.cholerae strain harboring a replicative-form CTX phage.

The term “recipient strain” used herein refers to a V. cholerae straininfected by a CTX phage produced from a donor.

The V. cholerae variant strain of the present invention may be increasedin expression of a TcpA protein, compared with a V. cholerae strainexpressing a wild-type toxT protein, and allows transcriptionalactivation of ctxAB expression, and thus the expression of cholera toxinmay be increased.

The V. cholerae variant strain of the present invention may be used as arecipient strain for CTX phage infection to improve transductionefficiency.

Therefore, the present invention provides a method of improving theinfection efficiency of a CTX phage, which includes transducing a V.cholerae strain with the recombinant plasmid for replicating a CTX phageof the present invention, or infecting the V. cholerae variant strain asa recipient strain using a V. cholerae strain harboring one or more CTXprophages selected from the group consisting of CTX-1, CTX-cla, CTX-2,CTX-env and CTX-O139 as a donor strain.

Generally, E1 Tor strains may not induce the production of cholera toxinunder general experimental conditions. Therefore, conditions forproducing the cholera toxin are induced using a method of reducing anoxygen partial pressure and increasing a CO₂ partial pressure under AKIconditions (biphasic culture, method of performing culture in astationary state for 16 hours and then further culture by changing theculture condition to shaking culture), but the cholera toxin is notproduced by single phase culture (condition for culturing stains only byshaking culture).

On the other hand, the V. cholerae variant strain of the presentinvention may produce the cholera toxin through single phase cultureunder conditions of 30 to 37° C. and pH 6 to 8.

The V. cholerae variant strain may be any V. cholerae strain expressingthe toxT protein of SEQ ID NO: 7 in which Tyr at amino acid 139 issubstituted with Phe through the point mutation of a toxT gene withoutlimitation, and may be, for example, a MG116025 (Matlab typeIII)-toxT^(Y139F) strain, B33-toxT^(Y139F) strain, A213-toxT^(Y139F)strain or IB4122-toxT^(Y139F) strain. In addition, a classical biotypestrain may also be used.

A medium that can be used according to the present invention has beendescribed above, and will be omitted to avoid excessive duplication.

The transformed strain is single phase-cultured, and then the choleratoxin is isolated and purified from the cell mass, culture or lysate ofthe strain, or the lysate of the culture, thereby obtaining the choleratoxin.

Hereinafter, the present invention will be described in detail withreference to examples. However, the following examples are merelyprovided to illustrate the present invention, and the scope of thepresent invention is not limited by the following examples.

EXAMPLES <Example 1> Construction of Plasmid-Based CTX Phage ReplicationSystem

By using a plasmid-based CTX phage replication system, the replicationof various CTX phages was performed under laboratory conditions. To thisend, CTX phages may be replicated from the plasmid-cloned CTX phagegenome. An E. coli- and V. cholerae-compatible recombinant plasmid wasconstructed by linking the replication origin of pUC18, the CTX-1 phagegenome in which total ctxA and a part of ctxB were substituted with akanamycin cassette and upstream and downstream non-coding sequences ofthe CTX phage (FIG. 1). CTX-1 was transformed with the recombinantplasmid, and CTX phage production from the plasmid-cloned CTX phagegenome was monitored through transduction of recipient strains. Cholerastrains used in the experiment are listed in Table 1.

The inventors disclosed strain PM8 in an article published in 2014 (Kim,E. J., et al. (2014) Molecular insights into the evolutionary pathway ofV. cholerae O1 atypical El Tor variants. PLoS Pathog. 10, e1004384),three types of host strains for the recombinant plasmid were PM14(N16961 derivative that has lost a lysogenic CTX-1 prophage in a N16961strain, which is a V. cholerae El Tor biotype strain, thus havingTLC:RS1 array), O395 (classical biotype strain) and A213 (U.S. GulfCoast strain harboring only the TLC element on chromosome 1). Thetransduction efficiency of CTX-1kan produced from plasmids using thehost was investigated (Table 2).

To construct a CTX phage plasmid shown in FIG. 1, the 674-bp replicationorigin DNA fragment of pUC18 was amplified by PCR using the primer pairof pUC18-ori-MluIF (5′-CCG CGC ACG CGT ATG TGA GCA AAA GGC-3′: SEQ IDNO: 8) and pUC18-ori-KpnIR (5′-CGC GCC GGT ACC CCC GTA GAA AAGATC-3′:SEQ ID NO: 9). The MluI restriction site (at nucleotide 245) ofzot, which is common in various CTX phage genomes, was used for plasmidconstruction. A zot-3′UTR fragment extending from the nucleotide 245 ofzot to the termination codon of rstR^(E1 Tor) of RS1 was amplified byPCR using the primer pair of zot-MluIF (5′-CGC GCG ACG CGT TTC TCT TTATCG ATG-3′: SEQ ID NO: 10) and 3′UTR-KpnIR (5′-CCG GCC GGT ACC CAA GACTCG CTA GCG-3′: SEQ ID NO: 11) from the PM8 strain containingTLC:CTX-1kan:RS1 on chromosome 1. The fragment was common to all CTXphages constructed in the present invention. The zot genes of CTX-1 andCTX-cla are different due to 14 SNPs. However, zot does not affect themorphology of CTX phages. A recombinant plasmid consisting of thereplication origin fragment of pUC18 and the zot-3′UTR fragment wasfirst constructed, and then the 5′ fragment was inserted into the MluIsite of the plasmid. Two 5′UTR-zot fragments of each CTX phage wereamplified using a common reverse transcription primer MluI (5′-CCG GCGACG CGT CCT TTC TCG CCC AGT GCC-3′: SEQ ID NO: 12) and a forward primer5′UTR MluIF (from the att site, 5′-CGC CCG ACG CGT TAG AGA CAA AAT GTTCCT-3′: SEQ ID NO: 13, the entire ig-1 sequence included) or 5′-119MluIF(from nucleotide-119 upstream of the termination codon of rstR of thelysogenic CTX genome, 5′-CGC CCG ACG CGT GCC TGT CCG CTG TGG-3′: SEQ IDNO: 14). The 5′ fragment of CTX-cla was amplified in classical strainCairo48, and the 5′ fragment of CTX-0139 was amplified in O139 strainAR1961537. Instead of 5′UTR MluIF, the 5′UTR-zot fragment of CTX-2 onchromosome 2 of strain B33 was amplified using the forward primerB33Ch2MluF (5′-CGC CCG ACG CGT ATG ATG TTT TTA TTC CAC-3′: SEQ ID NO:15).

An experiment was carried out to examine whether a CTX phage can betransferred to a recipient strain through transduction after thetransformation of a V. cholerae strain containing no CTX prophage or astrain containing no replicative-form CTX phage with the recombinantplasmid and the replication of the CTX phage from the strain. Briefly,0.5 mL of the supernatant of a donor strain culture grown overnight inthe presence of 20 ng/mL mitomycin C was mixed with 3×10⁸ CFU ofagglutinated recipient strains (grown at 30° C., pH 6.5 in LB). Thetransduction efficiency of each recipient strain was calculated by thenumber of transductants per 6×10⁸ CFU recipient cells per 1 mL of thesupernatant of the donor strain. Since the recipient strains havedifferent transduction efficiencies, the transduction efficiency of eachrecipient strain does not reflect the actual titer of the CTX phageproduced from the recipient strain. The transduction efficiency waspresented as the average of at least three independent experiments.

TABLE 1 Cholera strains and derivative strains thereof Gene structureCTX array in CTX array in Strain name chromosome 1 chromosome 2Description, genome information and reference V212-1 derivative V212-1TLC:RS1:CTX- CTX-2:CTX-2 Mutreja A, et al. (2011) Nature 477(7365):462465 1:RS1 PM8 TLC:CTX-1kan:RS1 CTX-2:CTX-2 Kim EJ, et al. (2014)PLoSPathog10(9): e1004384 PM9 TLC:RS1:CTX- CTX-2kan:CTX-2 Kim EJ, et al.(2014) PLoS Pathog 10(9): e1004384 1:RS1 N16961 Wave 1 El Tor strainsderivative N16961 TLC:CTX-1:RS1 No element AE003852/AE003853 HeidelbergJF, et al. (2000) Nature 406(6795): 477483 PM20 TLC:CTX-1- No elementPresent invention kan:RS1 PM27 TLC:RS1 No element Present invention PM14TLC:RS1 No element Kim EJ, et al. (2014) PLoSPathog10(9): e1004384MG116025 Wave 2 strains (Matlab type 3) derivative MG116025 TLC:RS1:CTX-No element ERS013135 1:RS1 Mutreja A, et al. (2011) Nature 477(7365):462465 MG116025- TLC:RS1:CTX- No element Present invention toxT-139Y1:RS1 PM30 TLC:RS1:CTX- No element PM30 is a strain in which CTX-2 isinserted 1:RS1:CTX-2-kan- downstream of the second RS1 of chromosome 1N2 through transduction of the MG116025 strain (having theTLC:RS1:CTX-1:RS1 array in chromosome 1 and nothing in chromosome 2),and thus has the TLC:RS1:CTX-1:RS1:CTX-2-kan configuration of chromosome1 and nothing on chromosome 2. PM31 TLC:RS1:CTX- CTX-2-kan-N2 Similar toPM30, PM31 is a strain in which CTX-2 is 1:RS1 inserted into chromosome2 through transduction of the MG116025 strain, and thus has theTLC:RS1:CTX- 1:RS1 array in chromosome 1 and CTX-2-kan on chromosome 2.PM25 TLC:CTX-1:RS1 No element Present invention PM32 TLC:CTX- No elementPM25 strain is a strain having TLC:CTX-1:RS1 array 1:RS1:CTX-2-kan- byremoving RS1 upstream of chromosome 1 of the N2 MG116025 strain andnothing on chromosome 2. PM32 strain is a strain in which CTX-2 isinserted into chromosome 1 through transduction of the PM25 strain, andthus has the TLC:CTX-1:RS1:CTX-2-kan array on chromosome 1 and nothingon chromosome 2. PM26 TLC:RS1:RS1 No element Present invention PM33TLC:RS1:RS1:CTX- No element PM33 is a strain in which CTX-1 is inserted1-kan-N2 downstream of RS1 of chromosome 1 through transduction of thePM26 strain, which has the TLC:RS1:RS1 array by removing CTX-1 fromchromosome 1 of MG116025 and has nothing on chromosome 2, and thus hasthe TLC:RS1:RS1:CTX-1- kan array and nothing on chromosome 2. PM28 TLCNo element Present invention PM34 TLC CTX-1kan-N2 PM34 is a strain inwhich CTX-1 is inserted into chromosome 2 through transduction of thePM28 strain, which has only TCL on chromosome 1 by removing both CTX-1and RS1 from chromosome 1 of MG116025 and nothing on chromosome 2, andthus has only TLC on chromosome 1 and CTX-1-kan on chromosome 2. PM35TLC:CTX-2-kan-N2 No element PM35 is a strain in which CTX-2 is insertedinto chromosome 1 through transduction of the PM28 strain, and thus hasthe TLC:CTX-2-kan array on chromosome 1 and nothing on chromosome 2.PM29 No TLC, no element No element Present invention PM36 CTX-2-kan-N2No element PM36 is a strain in which CTX-2 is inserted into chromosome 1through transduction of the PM29 strain, which has nothing on eitherchromosome 1 or 2 by removing all of TLC, RS1 and CTX-1 from MG116025,and thus has CTX-2-kan on chromosome 1 and nothing on chromosome 2. O395Classical biotype derivative O395 TLC:TrunCTX- CTX-cla CP000626/CP000627cla:CTX-cla Mutreja A, et al. (2011) Nature 477(7365): 462465 PM37TLC:TrunCTX- CTX-cla PM37 is a strain in which CTX-1 is insertedcla:CTX-cla:CTX-1- downstream of CTX-cla of chromosome 1 in a classicalkan-N2 biotype strain O395 (already having TLC:truncated CTX-cla:CTX-claon chromosome 1 and CTX-cla on chromosome 2). Classical biotype strainsare known to have only CTX-cla, and PM37 is a strain in which CTX- 1found only in El Tor strains is additionally inserted into a chromosome,and thus has TLC:TrunCTX- cla:CTX-cla:CTX-1-kan on chromosome 1 andCTX-cla on chromosome 2. B33 derivative Wave 2 El Tor strains B33 NoTLC, no element CTX-2:CTX-2 ACHZ00000000 Faruque SM, et al. (2007) ProcNatl Acad Sci USA 104(12): 51515156 PM38 No TLC:CTX-1-kan- CTX-2:CTX-2PM38 is an El Tor biotype strain in which CTX-1 is N2 inserted intochromosome 1 through transduction of the B33 strain which has nothing onchromosome 1 and the CTX-2:CTX-2 array on chromosome 2, and has CTX-1-kan on chromosome 1 and the CTX-2:CTX-2 array on chromosome 2. PM21 NoTLC, no element CTX-2 Present invention PM39 No TLC:CTX-1-kan- CTX-2PM39 is a strain in which CTX-1 is inserted into N2 chromosome 1 throughtransduction of PM21 strain which has only CTX-2 on chromosome 2 byremoving single CTX-2 from the CTX-2:CTX-2 array having chromosome 2 ofthe B33 strain and nothing on chromosome 1, and thus has CTX-1-kan onchromosome 1 and CTX-2 on chromosome 2. PM40 No TLC:CTX-2-kan- CTX-2PM40 is a strain which has CTX-2 on each of N2 chromosome 1 and 2 byinserting CTX-2 into chromosome 1 through transduction of PM21 strainPM22 No TLC, No element CTX-2kan:CTX-2 Present invention IB4122derivative IB4122-toxT- TLC:RS1:CTX-3 No element Mutreja A, et al.(2011) Nature 477(7365): 462465; 139Y Nguyen BM, et al. (2009) J ClinMicrobiol 47(5): 15681571 IB4122-toxT- TLC:RS1:CTX-3 No element Presentinvention 139F A213 US Gulf Coast strain, att+; A213 derivatives arestrains derivative constructed by inserting CTX phages into chromosomesthrough transduction of A213. A213 TLC No element ERS013191; A213derivatives are strains constructed by inserting CTX phages into thechromosomes through transduction of A213. A213-toxT- TLC No elementMutreja A, et al. (2011) Nature 477(7365): 462465 139Y A213-toxT- TLC Noelement Present invention 139F PM41 TLC:CTX-1-kan-N1 No element thestrain in which CTX-1 is inserted into chromosome 1 of an A213 strainPM42 TLC:CTX-1-kan- No element the strain in which a tandem repeat ofCTX-1 is inserted N1:CTX-1-cm-N1 into chromosome 1 of an A213 strainPM43 TLC:CTX-1-kan-N2 No element the strain in which CTX-1 is insertedinto chromosome 1 of an A213 strain PM44 TLC:CTX-1-kan- No element thestrain in which a tandem repeat of CTX-1 is inserted N2:CTX-1-cm-N1 intochromosome 1 of an A213 strain PM45 TLC CTX-1-kan-N2 the strain in whichCTX-1 is inserted into chromosome 2 of an A213 strain PM46TLC:CTX-1-cm-N1 No element the strain in which CTX-1 is inserted intochromosome 1 of an A213 strain PM47 TLC:CTX-1-cm- No element the strainin which a tandem repeat of CTX-1 is inserted N1:CTX-1-kan-N1 intochromosome 1 of an A213 strain PM48 TLC:CTX-1-cm- No element the strainin which a tandem repeat of CTX-1 is inserted N1:CTX-1-kan-N2 intochromosome 1 of an A213 strain PM49 TLC:CTX-1-cm- CTX-1-kan-N2 PM49 is astrain in which CTX-1 is further inserted into N1:CTX-1-kan-N2chromosome 2 through transduction of the PM48 strain, and thus has theTLC:CTX-1:CTX-1 array on chromosome 1 and CTX-1 on chromosome 2. PM50TLC:CTX-1-cm-N1 CTX-1-kan-N2 PM50 is a strain which has TC1:CTX-1 onchromosome 1 and CTX-1 on chromosome 2 by inserting CTX-1 into each ofchromosome 1 or 2 of A213strain. PM51 TLC:CTX-1-cm-N1 CTX-2-kan-N2 thestrain in which CTX-1 is inserted into chromosome 1 and CTX-2 isinserted into chromosome 2 in A213 PM52 TLC:CTX-2-kan-N2 No element thestrain in which CTX-2 is inserted into each of chromosome 1 andchromosome 2 through transduction of an A213 strain PM53 TLCCTX-2-kan-N2 the strain in which CTX-2 is inserted into each ofchromosome 1 and chromosome 2 through transduction of an A213 strain ThePM30~PM53 strains are derived from toxT-139F derivative of each parentstrain. pCTX-1-kan-N1a: constructed using a plasmid-based CTX phagereplication system pCTX-1-kan-N2b: constructed by recombination betweenCTX-1 and CTX-2 in V212-1 pCTX-1-cm-N1: constructed using aplasmid-based CTX phage replication system pCTX-1-cm-N2: constructedfrom PM48 pCTX-2-kan-N1: constructed using a plasmid-based CTX phagereplication system pCTX-2-kan-N2: constructed from PM22, which is a B33derivative N1: non-coding sequence derived from pCTX-1 N2: non-codingsequence derived from pCTX-2 or pCTX-cla

TABLE 2 Transduction efficiency of selected V. cholerae strains andplasmid-based replication of CTX phages Gene structure GeneratedTransduction Transduction Strain name Description Chromosome 1Chromosome 2 CTX phage recipient strain efficiency* PM20 N16961derivative TLC:CTX- No element CTX-1kan- O395 1 × 10⁵ 1kan:RS1 C1+MG116025 1 × 10² PM14 N16961 derivative TLC:RS1 No element no PM14-U1PM14 transformed TLC:RS1 No element CTX-1kan- O395 1 × 10⁵ with pUC-CTX-P++ 1kan PM14-U2 PM14 transformed TLC:RS1 No element CTX-2kan-P MG1160252 × 10 with pUC-CTX- 2kan PM14-U3 PM14 transformed TLC:RS1 No elementCTX-clakan-P MG116025 1 × 10² with pUC-CTX- clakan PM14-U4 PM14transformed TLC:RS1 No element CTX- O395 2 × 10² with pUC-CTX- O139kan-PMG116025 1 × 10 O139kan PM9 V212-1 derivative TLC:RS1:CTX-CTX-2kan:CTX-2 CTX-2kan- MG116025 1 × 10³ 1:RS2 C2+ PM22 B33 derivativeNo TLC, No CTX-2kan:CTX-2 CTX-2kan- MG116025 5 × 10³ element C2 O395Classical strain TLC:TrunCTX- CTX-cla no cla:CTX-cla O395-U1 O395transformed TLC:TrunCTX- CTX-cla CTX-1kan-P O395 5 × 10⁴ with pUC-CTX-cla:CTX-cla 1kan A213 US Gulf Coast TLC No element No strain A213-U1A213 transformed TLC No element CTX-1kan-P O395 3 × 10³ with pUC-CTX-1kan *Transduction efficiency was calculated as the number oftransductants per 6 × 10⁸ recipient cells per 1 mL of culturesupernatant of the donor strain. The data represent the average of atleast three independent experiments. C⁺: denoting CTX phage producedfrom lysogenic phage inserted into chromosome 1 or 2 P⁺⁺: denoting CTXphage produced from phage genome cloned in recombinant plasmid

As shown in Table 2, although there was a difference between hoststrains, when O395 was used as a recipient, ˜10⁵ transductants wereobtained per 1 mL of the donor strain supernatant. This was similar toCTX-1 phage production from the lysogenic CTX-1:RS2 array of N16961.This shows that the replication and propagation of CTX phages do notdepend on a host bacterial biotype. Since similar numbers oftransductants were obtained from plasmids containing the entireintergenic region 1 (ig-1) sequence and the last 119 nucleotides,respectively, the 119 nucleotides upstream of rstR were sufficient toinitiate CTX-1 replication in this system. Afterward, CTX phages wereproduced from the recombinant plasmid containing the last 119nucleotides of ig-1.

The replication origin of pUC18 was no longer present in the transformedCTX-1kan phage genome, and the DNA sequence of CTX-1kan produced fromthe recombinant plasmid was identical to CTX-1kan produced from thelysogenic CTX-1kan. PM20 is the strain in which CTX-1-kan phages can bereplicated from a prophage-type CTX genome, the CTX phage genomereplicated from the recombinant plasmid and the CTX phage genomereplicated from PM20 have the same base sequence, demonstrating that CTXphages replicated from the plasmid-cloned CTX phage genome andnaturally-replicated CTX phages are the same.

<Example 2> Replication of CTX-Cla, CTX-2 and CTX-O139 in Plasmid-BasedCTX Phage Replication System

Based on the confirmation that CTX-1 could be replicated from aplasmid-cloned CTX phage genome in V. cholerae, the replication of CTX-2and CTX-cla using the plasmid-based replication system could bepredicted. Recombinant plasmids containing CTX-clakan and CTX-2kangenomes were constructed by the method described in Example 1 totransform PM14. Due to phage immunity, it was expected that classicalbiotype strains could not be transduced by CTX-clakan or CTX-2kan PM14,and thus it was tested whether CTX-clakan and CTX-2kan phages producedfrom the pUC-CTX plasmids could be used to transduce various El Torbiotype strains. Recipient El Tor strains were prepared for transductionin the same manner as the classical strains (agglutinated, that is,grown in LB (pH 6.5) at 30° C.).

TABLE 3 Replication and transduction efficiency of CTX-cla or CTX-2 frompUC-CTX-2kan or pUC-CTX-clakan Primary transduction Secondarytransduction pCTX Transduction Transductant Transduction producedrecipient Transduction (transduction recipient Transduction Donor fromdonor strain efficiency pCTX) strain efficiency PM14-U2 pCTX-2kan-MG116025 2 × 10 MG116025 MG116025 2 × 10⁴ P* (pCTX-2kan-P) MG116025- 0toxTF139Y A213- 3 × 10³ toxTY139F IB4122 0 toxTY139F PM14-U3 pCTX-MG116025- 0 clakan-P⁺ toxTF139Y MG116025 1 × 10² MG116025 MG116025 3 ×10⁴ (pCTX-clakan-P) MG116025- 0 toxTF139Y A213- 2 × 10⁴ toxTY139FIB4122- 0 toxTY139F PM9 pCTX-2kan- MG116025- 0 VL⁺⁺ toxTF139Y MG116025 1× 10³ MG116025 (pCTX- MG116025 2 × 10⁷ 2kan-VL) MG116025- 0 toxTF139YA213- 1 × 10⁶ toxTY139F IB4122- 1 × 10³ toxTY139F MG116025- 0 toxTF139Y*pCTX-2kan-P: pCTX-2kan is produced from plasmid pUC-CTX-2kan.⁺pCTX-clakan-P: pCTX-clakan is produced from plasmidpUC-CTX-clakan.⁺⁺pCTX-2kan-VL: pCTX-2kan is produced from lysogenic CTX-2 in PM9, whichis a derivative of a V212-1 strain. MG116025-toxTF139Y: MG116025 strainin which F at amino acid 139 is substituted with Y through the pointmutation of the toxT gene

As shown in Table 3, no El Tor strains were transduced by CTX-clakan andCTX-2kan except Wave 2 El Tor strain MG116025. This strain wastransduced by CTX-clakan and CTX-2kan phages replicated using theplasmid-based replication system. Approximately 100 and 20 transductantswere obtained from the plasmid-cloned CTX-clakan and CTX-2kan genomes,respectively. The replication of CTX-clakan and CTX-2kan was confirmedby secondary transduction. Based on these results, the replication ofCTX-2 and CTX-cla was confirmed with a suitable recipient strain,demonstrating the proper operation of the plasmid-based CTX phagereplication system. Similarly, CTX-O139kan phages were also producedusing the plasmid-based replication system.

The replication of CTX-2 from a tandem repeat of lysogenic CTX-2 presenton chromosome 2 of a Wave 2 El Tor (Tor 2) strain was also demonstrated.PM9 and PM22, and V212-1 and B33 were constructed as describedpreviously. Approximately 10³ transductants were obtained throughprimary transduction of PM9 and PM22. Secondary transduction from thetransductants containing replicative forms of pCTX-clakan and pCTX-2kanto CTX-cla and CTX-2 was further demonstrated. Since no classical straincontaining the CTX-cla:RS1 array or the tandem repeat of CTX-cla hasbeen reported yet, the replication of lysogenic CTX-cla in the classicalbiotype strains could not be proved.

<Example 3> Transduction of CTX Phage Produced from El Tor Strain N16961

To identify genetic changes facilitating the susceptibility of theMG116025 strain to a CTX phage, genetic changes in the tcp gene clusterof MG116025, which was caused by TCP-mediated CTX phage infection, wasexamined.

Compared with other El Tor strains, two SNPs specific to the tcp genecluster of MG116025 were identified from genome sequencing data. Ala56(C269) of tcpA is changed to Asp (A269), and Tyr139 (A416) of toxT issubstituted with Phe (T416) in MG116025. When Asp56 of tcpA of MG116025is switched to Ala, the transduction efficiency was not changed orincreased by up to 10 fold depending on a CTX phage, indicating thatthis change is not significant to the transduction-competent phenotypeof MG116025. When Phe139 of toxT of MG116025 was substituted with Tyr,the transduction efficiency was decreased, suggesting that the SNPsmediate the ability of MG116025 transformed by CTX phages (Table 3-7).

Subsequently, the toxT allele (Tyr139) of two El Tor strains which werenot transduced by CTX phages such as A213 and IB4122 (Wave 3 El Torstrain containing RS1:CTX-3 on chromosome 1) was substituted with thetoxT allele (Phe139) of MG116025, and the ability of the strains to betransduced by CTX phages was examined.

To this end, from MG116025 and N16961 strains, a DNA fragment from 50nucleotides upstream of the ATG initiation codon to nucleotide 793 oftoxT was amplified by PCR using toxT-XbaIF (5′-CCG GCC TCT AGA TAC GTGGAT GGC TCT CTG CG-3′: SEQ ID NO: 16) and toxTSacIR (5′-CCG GCC GAG CTCCAC TTG GTG CTA CAT TCA-3′: SEQ ID NO: 17) primers, and inserted intosuicide plasmid pCVD442. The SNP position at nucleotide 416 (A416 ofN16961 and T416 of MG116025) was placed in the center of the fragment.The MG116025 toxT 139Phe allele was replaced with the toxT 139Tyr alleleby an allelic exchange method, and similarly, IB4122 and A213 toxT139Tyr alleles were replaced with the toxT 139Phe allele.

A213-toxT^(Y139F) and IB4122-toxT^(Y139F) were transduced by CTX phageswhen grown in LB (pH 6.5) at 30° C. (Tables 4 to 7), and A213 and IB4122were not transduced by a CTX phage.

TABLE 4 Replication and production of CTX-1 Primary transductionSecondary transduction pCTX Transduction Transduction produced recipientTransduction Transductant recipient Transduction Donor from donor strainefficiency (transduced pCTX) strain efficiency PM14-U1 pCTX-1kan- O395 1× 10⁵ O395 (pCTX-1kan- O395 5 × 10⁷ P* P) MG116025 3 × 10⁵ MG116025- 0toxTF139Y A213- 1 × 10⁵ toxTY139F IB4122 5 × 10  toxTY139F PM14-U1pCTX-1kan- MG116025 1 × 10² MG116025 (pCTX- O395 5 × 10⁶ P* 1kan-P)MG116025 1 × 10³ MG116025- 0 toxTF139Y A213- 1 × 10  toxTY139F IB4122 0toxTY139F PM14-U1 pCTX-1kan- MG116025- 0 P* toxTF139Y PM14-U1 pCTX-1kan-A213- 1 × 10⁴ A213- O395 5 × 10⁴ P* toxTY139F toxTY139F (pCTX- MG1160252 × 10  1kan-p) MG116025- 0 toxTF139Y A213- 2 × 10² toxTY139F IB4122 0toxTY139F PM20 pCTX-1kan- IB4122- 1 × 10² B4122 L+ toxTY139F toxTY139F(pCTX- 1kan-P) PM20 pCTX-1kan- O395 1 × 10⁵ O395 (pCTX-1kan- O395 3 ×10⁷ L+ L) MG116025 3 × 10⁵ MG116025- 0 toxTF139Y A213- 1 × 10⁵ toxTY139FIB4122 1 × 10³ toxTY139F PM20 pCTX-1kan- MG116025 1 × 10² MG116025(pCTX- O395 5 × 10⁴ L+ 1kan-L) MG116025 5 × 10  MG116025- 0 toxTF139YA213- 1 × 10² toxTY139F IB4122 10  toxTY139F PM20 pCTX-1kan- MG116025- 0L+ toxTF139Y PM20 pCTX-1kan- A213- 1 × 10⁴ A213- O395 2 × 10⁵ L+toxTY139F toxTY139F (pCTX- MG116025 10  1kan-L) MG116025- 0 toxTF139YA213- 1 × 10² toxTY139F IB4122 0 toxTY139F PM20 pCTX-1kan- IB4122 10  L+toxTY139F *pCTX-1kan-P: pCTX-1kan is produced from plasmid pUC-CTX-1kan.+pCTX-1kan-L: pCTX-1kan is produced from lysogenic CTX prophage.

TABLE 5 Replication and production of CTX-2 Primary transductionSecondary transduction pCTX Transduction produced Transductiontransduction Transductant recipient transduction Donor from donorrecipient strain efficiency (transduced pCTX) strain efficiency PM14-U2pCTX-2kan- MG116025 2 × 10 MG116025 (pCTX- MG116025 2 × 10⁴ P* 2kan-P)MG116025- 0 toxTF139Y A213- 3 × 10³ toxTY139F IB4122 0 toxTY139F O395 0PM14-U2 pCTX-2kan- MG116025- 0 P* toxTF139Y PM14-U2 pCTX-2kan- A213- 10 P* toxTY139F PM14-U2 pCTX-2kan- IB4122 0 P* toxTY139F PM14-U2 pCTX-2kan-O395 0 P* PM22 pCTX-2kan- MG116025 5 × 10³ MG116025 (pCTX- MG116025 1 ×10⁷ BL+ 2kan-BL) MG116025- 10  toxTF139Y A213- 4 × 10⁶ toxTY139F IB41221 × 10² toxTY139F O395 300++ PM22 pCTX-2kan- MG116025- 0 BL+ toxTF139YPM22 pCTX-2kan- A213- 5 × 10² BL+ toxTY139F PM22 pCTX-2kan- IB4122 10 BL+ toxTY139F PM22 pCTX-2kan- O395 0 BL+ PM9 pCTX-2kan- MG116025 1 × 10³MG116025 (pCTX- MG116025 2 × 10⁷ VL+++ 2kan-VL) MG116025- 0 toxTF139YA213- 1 × 10⁶ toxTY139F IB4122 1 × 10³ toxTY139F O395 200++ PM9pCTX-2kan- MG116025- 0 BL+ toxTF139Y PM9 pCTX-2kan- A213- 1 × 10³A213-toxTY139F MG116025 2 × 10³ BL+ toxTY139F (pCTX-2kan-VL) MG116025- 0toxTF139Y A213- 2 × 10³ toxTY139F IB4122 0 toxTY139F O395 0 PM9pCTX-2kan- IB4122 10  IB4122 BL+ toxTY139F toxTY139F (pCTX- 2kan-VL)*pCTX-2kan-P: pCTX-2kan is produced from plasmid pUC-CTX-2kan.+pCTX-2kan-BL: pCTX-2kan is produced from lysogenic CTX-2 in PM strain,which is a derivative of B33 strain. ++O395 transductants are producedby CTX-2kan (⅓ of transductants) or CTX phage (⅔ of transductants),which contains rstRE1 Tor generated by interstrand recombination betweenCTX-2kan and lysogenic CTX-1 in MG116025 +++pCTX-2kan-VL: pCTX-2kan isproduced from lysogenic CTX-2 in PM9, which is a derivative of V212-1strain.

TABLE 6 Replication and production of CTX-cla Primary transduction pCTXTransductant Secondary transduction produced from Transductiontransduction (transduced Transduction Transduction Donor donor recipientstrain efficiency pCTX) recipient strain efficiency PM14-U3 pCTX-clakan-MG116025  1 × 10² MG116025 MG116025 3 × 10⁴ P* (pCTX-clakan- MG116025- 0P) toxT^(F139Y) A213-toxT^(Y139F) 2 × 10⁴ IB4122 0 toxT^(Y139F) O395 0MG116025- 0 toxT^(F139Y) A213-toxT^(Y139F) 4 × 10 A213-toxT^(Y139F)(pCTX- clakan-P) IB4122 3 × 10 IB4122 toxT^(Y139F)toxT^(Y139F) (pCTX- clakan-P) O395 0 *pCTX-clakan-P: pCTX-clakan isproduced from plasmid pUC-CTX-clakan.

TABLE 7 Replication and production of CTX-O139 Primary transduction pCTXTransduction Secondary transduction produced from recipient TransductionTransductant Transduction Transduction Donor donor strain efficiency(transduced pCTX) recipient strain efficiency PM14-U4 pCTX- O395 2 × 10²O395 (pCTX- O395 1 × 10⁶ O139kan-P* O139kan-P) MG116025 5 × 10³MG116025- 0 toxT^(F139Y) A213- 1 × 10⁶ toxT^(Y139F) IB4122 1 × 10²toxT^(Y139F) PM14-U4 pCTX- MG116025 10 MG116025 (pCTX- O395 5 × 10⁶O139kan-P* O139kan-P) MG116025 3 × 10⁴ MG116025- 0 toxT^(F139Y) A213- 3× 10³ toxT^(Y139F) IB4122 4 × 10² toxT^(Y139F) PM14-U4 pCTX- MG116025- 0 O139kan-P* toxT^(F139Y) PM14-U4 pCTX- A213- 30 A213- O139kan-P*toxT^(Y139F) toxT^(Y139F (pCTX-) O139kan-P) PM14-U4 pCTX- IB4122 20IB4122 O139kan-P* toxT^(Y139F) toxT^(Y139F) (pCTX- O139kan-P)*pCTX-O139kan-P: pCTX-clakan is produced from plasmid pUC-CTX-O139kan.

The CTX phage infection in V. cholerae strains was mediated by TCP as aCTX phage recipient. Phenotypes enabling transduction of strainsharboring the toxT 139Phe allele may be mediated by the high expressionof TCP. TcpA expression was assessed by western blotting to monitor aTCP level.

For western blotting, bacterial strains were grown overnight in a LBmedium at 30° C., subcultured in a LB medium (pH 6.5) at 30° C. or in LBdiluted 1:100 at 37° C., and grown overnight again. Cells were pelleted,resuspended in 1×sample buffer, and boiled for 5 minutes. Approximately5×10⁷ cells were loaded onto each lane in an SDS-PAGE gel. Proteins weretransferred to a Protran nitrocellulose membrane (GE Healthcare) andprobed with rabbit anti-CT (Sigma) or rabbit anti-TcpA (W. F. Wade,Dartmouth University, Hanover, N.H.).

As shown in FIGS. 2 and 3, while El Tor strains containing the toxT139Phe allele expressed more TcpA, TcpA was not detected in strainscontaining the toxT 139Tyr allele. These results show that the toxT139Phe allele in the El Tor strains up-regulates TCP so that CTX phageinfection in bacteria may increase. Although containing the toxT133Tyrallele, high TcpA expression in the O395 strain was observed. Theseresults have been previously known and are known as characteristics ofclassical biotype strains and El Tor biotype strains.

<Example 4> Replication and Transduction of CTX Phages

Primary and secondary transduction of various CTX phages was evaluatedfor O395, MG116025, A213-toxT^(Y139F) and IB4122-toxT^(Y139F)recipients. Briefly, El Tor strains having the toxT-Phe139 allele may betransduced by CTX phages, but efficiencies vary depending on a strain.Compared with a lysogenic or plasmid-cloned CTX phage genome (primarytransduction), the production of CTX phages was ˜10²-fold (CTX-1 andCTX-cla) to 10⁴-fold (CTX-2 and CTX-O139) higher by replicative-formpCTXs (secondary transduction). Electron microscopy images of CTX-1kan,CTX-2kan and CTX-O139kan were obtained when the viral titer exceeded 10⁶particles per mL (FIGS. 4A-4C). In the transductants of various CTXphages, the integration of pCTXs into chromosome 1 or 2 was confirmed.

CTX-1: When O395, A213-toxT^(Y139F) and MG116025 (andIB4122-toxT^(Y139F)) were recipients, approximately 10⁵, 10⁴ and 10²transductants were obtained from PM14-U1 (PM14 transformed withpUC-CTX-1kan) (Table 4). When O395 was a recipient, secondarytransduction efficiency by pCTX-1kan in the O395 transductant increasedup to 5×10⁷, indicating that ˜10% of the recipient cells were transducedby CTX-1kan. When MG116025 was a recipient, 10⁴ transductants may beobtained with the same CTX-1kan phage titer because of the phageimmunity by lysogenic CTX-1 and RS1 (described below). The replicationefficiency of pCTX-1kan also varied depending on a host strain. 5×10⁷transductants were obtained from O395 (pCTX-1kan), whereas fewertransductants (5×10⁶ and 5×10⁴, respectively) were produced fromMG116025 (pCTX-1kan) and A213-toxT^(Y139F) (pCTX-1kan). The replicationefficiency of the CTX-1kan phages produced from lysogenic CTX-1 (PM20strain) was similar to that from the pUC-CTX-1kan plasmid. Electronmicroscopy images of the CTX-1kan phages were obtained from the culturesupernatant of O395 (pCTX-1kan) as shown in FIG. 4A.

CTX-2: pCTX-2kan replicated from a pUC-CTX-2kan plasmid was transferredto MG116025 and IB4122-toxT^(Y139F) at an efficiency of 20 and 10transductants, respectively (Table 5). Approximately 2×10⁴ transductantswere obtained in secondary transduction from the primary transductantMG116025, which harbored pCTX-2kan-P. The replication efficiency oflysogenic CTX-2 varied depending on a recipient strain. Approximately5×10³ transductants were obtained from PM22 (B33 derivative), and 10³transductants were obtained from PM9 (V212-1 derivative). No CTX phagewas produced from the second CTX-2 prophage of V212-1 (PM10).

A213-toxT^(Y139F) was also transduced at a slightly lower efficiencythan MG116025, but the transduction efficiency of IB4122-toxT^(Y139F)was significantly lower. O395 classical biotype strains were alsotransduced by the supernatant of PM9, but pCTX transferred to O395contained rstR^(E1 Tor), which was expected to be generated by therecombination between CTX-2kan produced from the plasmid and CTX-1 onchromosome 1 and caused by the immunity imparted by rstR^(cla) inlysogenic CTX-cla. Perhaps, since classical biotype strains already havethe rstR^(cla) gene, the efficiency of transducing CTX-2 containingrstR^(cla) may be expected to be reduced.

The secondary transduction efficiency of pCTX-2kan produced from PM9 orPM22 exceeded 10⁶ when MG116025 was a recipient. A213-toxT^(Y139F) andIB4122-toxT^(Y139F) were transduced by a high CTX-2kan phage titer. Theclassical strain O395 was not transduced by a low CTX-2kan phage titer,but by a high CTX-2kan phage titer. Among several hundred transductants,approximately ⅔ of O395 transductants were transduced by a CTX phagecontaining rstR^(E1 Tor), and ⅓ were transduced by CTX-2. Electronmicroscopy images of CTX-2kan phages in the culture supernatant ofMG116025 (pCTX-2kan) were taken (FIG. 4B).

CTX-cla: The transduction efficiency of CTX-clakan from pUC-CTX-clakaninto MG116025 was 10² transductants, and 10⁴ transductants were formedin the secondary transduction (Table 6). A213-toxT^(Y139F) andIB4122-toxT^(Y139F) were also transduced by CTX-clakan in the primaryand secondary transduction at a slightly lower efficiency than MG116025.Due to the low CTX-clakan titer, electron microscopy images ofCTX-clakan phages were not obtained.

CTX-O139: The replication and production of infectious CTX-O139 from alysogenic CTX-O139 genome were reported, and thus CTX-O139 replicationfrom plasmid-cloned CTX-0139 was demonstrated. When O395 and MG116025were recipients, ˜200 and 10 transductants may transfer CTX-O139kanphages which were produced in PM14-U4 by transduction, respectively(Table 7). The secondary transduction efficiency increased up to 10⁶transductants, and electron microscopy images of CTX-O139kan phages weretaken (FIG. 4C). A213-toxT^(Y139F) and IB4122-toxT^(Y139F) were alsotransduced by a high CTX-O139kan phage titer.

<Example 5> Phage Immunity

To measure the degree of inhibition of superinfection of the CTX-1 phageby lysogenic CTX-1 and RS1, a set of isogenic strains of MG116025 wasconstructed by stepwise removal of CTX-1 and RS1 (strains PM25˜PM29). Tothis end, MG116025, PM25 (TLC:CTX-1:RS1), PM26 (TLC:RS1:RS1), PM27(TLC:RS1), PM28 (TLC) and PM29 (no TLC, no element) were constructed bystepwise removal of CTX-1 and RS1 on chromosome 1. pCVDrstRET whichcontained a DNA fragment including rstR^(E1 Tor) (339 bp) and the first226 bp of rstA was inserted independently into rstR^(E1 Tor) of CTX-1and RS1 of MG116025. The removal of CTX-1 and RS1 was verified byanalyzing the genetic structure of strains selected from LB platescontaining 15% (wt/vol) sucrose.

In addition, to measure phage immunity, the inhibition of CTX-1superinfection by lysogenic CTX-1 or RS1 was monitored by measuring thetransduction efficiency of isogenic strains of MG116025. MG116025 andPM25˜PM29 were transfected by pCTX-1kan phages produced from O395transductants by pCTX-1kan. The immunity against CTX-2 was tested in thesame strain as the pCTX-2kan phage produced from the MG116025transductant by pCTX-2kan.

PM28 and PM29, which do not contain CTX-1 or RS1, were transduced byCTX-1 at ˜80% efficiency; when 6×10⁸ recipient cells were used, 5×10⁸transductants were obtained (Table 8), which was 10-fold higher than theclassical strain O395. When PM27 containing one RS1 (thus, 1rstR^(El Tor)) was used as a recipient, transduction efficiency wasalmost the same. The transduction efficiency was reduced by ˜10⁻³-foldin PM26 and PM25 strains as well as the MG116025 strain. These resultsshow that CTX-1 superinfection was limited by rstR^(E1 Tor) of lysogenicCTX-1 or RS1. However, one rstR is not sufficient to inhibitsuperinfection, and at least two rstR genes are needed to inhibit thesuperinfection. While the superinfection of the CTX-1kan phage wasrestricted by the preexisting RS1 and CTX-1, the infection ofrstR^(cla)-containing CTX-2kan was not influenced by rstR^(E1 Tor)(heteroimmunity).

TABLE 8 Inhibition of superinfection of CTX-1kan phage by residentrstR^(El Tor) in lysogenic CTX-1 and RS1 No. of transductants No. oftransductants Recipient by CTX-1kan by CTX-2kan MG116025 (TLC:RS1:CTX- 3× 10⁵ 5 × 10⁷ 1:RS1) PM25 (TLC:CTX-1:RS1) 3 × 10⁵ 4 × 10⁷ PM26(TLC:RS1:RS1) 5 × 10⁵ 5 × 10⁷ PM27 (TLC:RS1) 5 × 10⁸ 6 × 10⁷ PM28 (TLC)5 × 10⁸ 5 × 10⁷ PM29 (No TLC, No element) 5 × 10⁸ 5 × 10⁷

<Example 6> Production of toxT and Cholera Toxin

ToxT is a 32-kDa AraC family transcriptional activator. ToxT contains aconserved C-terminal DNA-binding domain including 100 amino acids, andits 176-amino acid N terminus is a dimerized domain. ToxT directlycontrols the expression of the tcp gene cluster and ctxAB. The toxT-F139allele of El Tor strains up-regulated TCP at 30° C., pH 6.5, enablingthe bacteria to be transduced by CTX phages. This result indicates thatthe toxT-F139 allele increases the expression of cholera toxin. Theproduction of cholera toxin in V. cholerae strains was assessed byWestern blotting (FIGS. 5-6B).

While no cholera toxin was produced in 37° C. culture, production wasobserved at 30° C., pH 6.5 in O395, MG116025 and IB4122-toxT^(Y139F).The expression of cholera toxin was shut off in MG116025-toxT^(F139Y),demonstrating that SNP in toxT mediates the increased expression of TCPand cholera toxin of El Tor biotypes under laboratory conditions.

In addition, since El Tor strains may not induce toxin production undergeneral laboratory conditions, conditions for producing a toxin wereinduced using a method of lowering an oxygen partial pressure andincreasing a CO₂ partial pressure under AKI conditions (biphasicculture, method of culturing for 16 hours in a stationary state and thenculturing under different culture conditions), but no toxin was producedin single phase culture (condition for culturing a strain only inshaking culture). However, the results show that the cholera toxin canalso be produced in toxT variant strains in single phase culture.

<Example 7> Construction of E1 Tor Strain Simultaneously HavingPlurality of CTX Phages

Since the replication and propagation methods of CTX-2 and CTX-cla,which were used in the above-describe examples, had been setup inlaboratories, strains having more various CTX phages and arrays weredesigned and constructed. The CTX phages were transferred bytransduction and inserted, and a plasmid form was removed, such that ElTor strains having both CTX-1 and CTX-2 were constructed. In addition toEl Tor biotype strains, a non-typical classical strain in which CTX-1was inserted into a classical biotype strain was also constructed. Thecholera strains used in this example have been listed in Table 1.

(Construction of pCTX Phages)

pCTX-1-kan-N1 is authentic pCTX-1 having a non-coding sequence which isthe same as pCTX-1, and having a kanamycin cassette which is substitutedfor ctxA and the first 166 bp of ctxB (Table 1). pCTX-1-kan-N1 may begenerated by replication of lysogenic CTX-1-kan in a strain such asPM20, or from a plasmid-based CTX phage replication system.pCTX-1-kan-N2 may be constructed by recombination between CTX-1 andCTX-2 in a PM9 strain. pCTX-1-kan-N2 has a non-coding sequence ofpCTX-2/pCTX-cla. pCTX-1-cm-N1, like CTX-1-kan-N1, may be generated usinga plasmid-based CTX phage replication system. In this system, achloramphenicol cassette is substituted instead of the kanamycincassette. pCTX-1-cm-N2 is generated from PM48.

pCTX-cla-kan-N1 has the non-coding sequence of pCTX-1, and is generatedfrom a plasmid-based CTX phage replication system. pCTX-2-kan-N1 isgenerated from a plasmid-based CTX phage replication system.pCTX-2-kan-N1 has the non-coding sequence of pCTX-1. pCTX-2-kan-N2 isgenerated from the CTX-2 tandem repeat of PM22 generated in B33.

The pCTX phages generated from a plasmid-based CTX phage replicationsystem or a lysogenic CTX prophage were maintained by transduction ofsuitable recipient strains for maintenance, that is, transduction of anO395 classical strain using pCTX-1 or a PM27-toxT-139F strain usingpCTX-2, to produce more progeny phages, and used for secondarytransduction.

(Construction of MG116025 and B33 Derivative Strains)

Isogenic strains of MG116025 and PM25 to PM29 were constructed bystepwise removal of CTX-1 and RS1. PM21, which is a derivative strain ofB33, contained only one CTX-2 on chromosome 2, which was similar to PM6.These strains were used to construct strains having various CTX arrays.

(Construction of CTX Phage Transduction Recipient Strains)

When cultured under agglutination conditions (LB, pH6.5, 30° C.), apCTX-1 phage was inserted into a classical biotype strain O395. When ElTor biotype strains harbored the toxT-139F allele, the strains weretransduced by CTX phages. Strains such as A213, B33, MG116025, etc. wereprepared to serve as a transduction recipient by substitution withtoxT-139F.

(Insertion of CTX Phages into Cholera Strains)

The CTX phages replicated in a CTX phage donor strain are transferredthrough transduction to a recipient strain according to a standardprotocol. The insertion of pCTX into chromosome 1 or chromosome 2 andthe final CTX array were confirmed by various PCR combinations. pCTX wasinserted into the chromosome, and a pCTX-cured strain was screened.

<Experimental Example 1> CTX Phages and Non-Coding Sequences

The non-coding sequences (the sequence between ctxB and rstR) of pCTX-1and pCTX-cla differ from each other. The difference in base sequence ofthe non-coding sequences of pCTX-1 and pCTX-cla determines theorientation of inserting CTX phages into either chromosome 1 orchromosome 2 of cholera strains. When a phage has the non-codingsequence of pCTX-1, it has been known that the sequence is inserted onlyinto chromosome 1, and when a phage has the non-coding sequence ofpCTX-cla, the sequence may be inserted into both chromosome 1 andchromosome 2. The non-coding sequences of pCTX-1 and pCTX-2 may beinterchangeable. CTX-1-kan-N1 contains the non-coding sequence ofauthentic pCTX-1, and CTX-1-kan-N2 contains the non-coding sequence ofpCTX-cla. Therefore, pCTX-1-kan-N2 can be expected to integrate intoboth chromosome 1 and chromosome 2, and PM34 (also PM45, 49 and 50) wasindeed constructed by inserting pCTX-1-kan-N2 into chromosome 2.

The non-coding sequence of pCTX is derived from RS1 downstream of a CTXphage during the replication of CTX prophages or a different prophage.Since most El Tor strains have a CTX-1:CTX-1 array or CTX-1:RS1 array,pCTX-1 has the non-coding sequence of authentic CTX-1. However, if thereare CTX-1:CTX-2 and CTX-1:CTX-cla arrays, CTX-1 having the non-codingsequence of CTX-cla may be generated while CTX-1 is replicated.Therefore, the pCTX-1-N2 phage having both the pCTX-1 genome and thenon-coding sequence of CTX-cla may be present like pCTX-1-N1. When thepCTX-1-N2 phage is inserted downstream of CTX-1 of a different strain,another pCTX-1-N2 phage may be generated. In this study, pCTX-1-cm-N2was actually generated from PM48 (Table 1).

<Experimental Example 2> Recombination Between pCTX and Prophage

A mechanism of generating mosaic CTX phages by recombination between twodifferent CTX phages present on different chromosomes in a singlecholera strain had been previously demonstrated. Similarly,recombination between pCTX and a prophage could be possible.pCTX-1-kan-N1 is transferred to a B33-toxT-139F strain, therebygenerating B33 (pCTX-1-kan-N1), and the phage progeny pCTX-1-kan-N1generated from this strain may be transduced to O395 to avoid phageimmunity. However, the recombination between pCTX-1-kan-N1 and a CTX-2prophage may occur in B33 (pCTX-1-kan-N1), and when the recombinationbetween rstA and a non-coding sequence, which are present at both sidesof rstR of each CTX phage, occurs, pCTX-2-kan-N1 may be produced (FIG.7A). The pCTX-2-kan-N1 produced thereby may be transferred to an El Torstrain such as MG116025. Indeed, pCTX-2-kan-N1 transferred to MG116025through transduction was able to be identified. These results show thatpCTX-2 can be generated by transducing a classical strain harboring atandem of the CTX-cla prophage with pCTX-1 (FIG. 7B). It is alsopossible to have recombination between pCTX-1 and pCTX-cla.

<Experimental Example 3> Construction of Classical Biotype StrainHarboring CTX-1

While atypical El Tor strains (Wave 2) harboring CTX-2 have been found,no classical strain having CTX-1 containing rstR^(El Tor) andctxB^(El Tor) has been found. Classical biotype strains are transducedby CTX-1, and while pCTX-1 is maintained in a classical biotype strain,a strain prepared by inserting CTX-1 into the chromosome of a classicalbiotype strain has not been found yet. As shown in FIG. 8, a strain wasconstructed by inserting pCTX-1-kan-N2 into chromosome 1 of O395, whichis a classical biotype strain (PM37). This result shows that atypicalclassical strain might be present in nature.

<Experimental Example 4> Design and Construction of Various El TorStrains

Toxigenic El Tor-type strains are generated by the integration of TLC,CTX-1 and RS1 in the dif1 base sequence of chromosome 1. In addition, ithas been previously reported that strains that do not generate a toxinare generated by removing CTX-1 and RS1 from El Tor biotype strains. Forexample, the MG116025 strain has the TLC:RS1:CTX-1:RS1 array onchromosome 1, and a process of generating isogenic strains of theMG116025 strain, which are generated by stepwise removal of RS1, CTX-1and even TLC (PM25˜PM29), has been previously reported. In this study,various types of pCTXs are transferred to MG116025 and strains generatedtherefrom, El Tor strains such as B33, A213, etc., thereby constructingstrains having various CTX arrays.

MG116025 and derivative strains thereof: As shown in FIG. 9, MG116025and derivative strains thereof are strains which already contain theCTX-1 phage and RS1 on their chromosomes. It was demonstrated that novelstrains having various CTX arrays can be substantially designed andconstructed by inserting CTX-1 and CTX-2 phages into the chromosomes ofMG116025 and its derivative strains.

PM30 and PM31 strains are strains generated by inserting CTX-2 intochromosome 1 and chromosome 2 of MG116025 (FIG. 8, Table 1). Similarly,PM32 harboring TLC:CTX-1:RS1:CTX-2-kan may be generated from a PM25strain. PM33 harboring TLC:RS1:RS1:CTX-1 on chromosome 1 may begenerated by inserting pCTX-1-kan-N2 into PM26. PM28 is a strainharboring only TLC on chromosome 1, and PM34 may be generated byinserting pCTX-1-kan-N2 into chromosome 2 of PM28. In addition, PM35 isa strain generated by inserting CTX-2 into chromosome 1 of PM28. Theconstruction of strains having more various arrays from a strainharboring only TLC will be described with strains generated from an A213strain in further detail below. A PM36 strain was constructed byinserting CTX-2-kan-N2 into chromosome 1 of PM29, which is a strainhaving no TLC. These results show that it is possible to additionallyintroduce a CTX phage into strains conventionally harboring CTX, RS1,etc., or insert a novel CTX phage after removal of preexisting CTX andRS1.

B33 and derivative strains thereof: As shown in FIG. 10, B33 is a Wave 2atypical El Tor strain that harbors no TLC on chromosome 1 and twotandem repeats of CTX-2 on chromosome 2. CTX-1 may be inserted intochromosome 1 of B33, thereby generating a PM38 strain, which harborsdifferent types of CTX phages on respective chromosomes. PM21 is astrain generated by removing one CTX-2 from B33, and PM39 and PM40 maybe generated by inserting CTX-1 and CTX-2 into chromosome 1 of PM21,respectively.

A213 and derivative strains thereof: A213 is a strain which isclassified as a US Gulf Coast strain or a pre-seventh pandemic strainand does not harbors CTX, which might be lost during isolation orstorage. This study has more focused on constructing various strainsfrom A213, which is to demonstrate a principle for a method ofconstructing a strain having various CTX phage arrays since A213 is astrain harboring only TLC.

As shown in FIG. 11, a strain harboring only one CTX-1 or CTX-2 on eachchromosome was constructed, and a strain harboring a tandem repeat ofCTX-1 on a chromosome (PM43) was also constructed. A strain harboringCTX-1 on both chromosomes (PM50), and a strain harboring a tandem repeatof CTX-1 on chromosome 1 and CTX-1 on chromosome 2 (PM49) were alsoconstructed. In addition, a strain harboring CTX-1 on chromosome 1 andCTX-2 on chromosome 2 (PM51) and a strain harboring solitary CTX-2 onchromosome 2 (PM53) were constructed.

The present invention can be applied in the field of producing a choleravaccine. In addition, the present invention can be applied in order toproduce cholera toxin or a toxoid.

What is claimed is:
 1. A Vibrio cholerae variant strain, comprising:expressing a toxT protein having the amino acid sequence of SEQ ID NO: 7in which Tyr at amino acid 139 of SEQ ID NO: 7 is substituted with Phethrough the point mutation of a toxT gene.
 2. The Vibrio choleraevariant strain according to claim 1, wherein the Vibrio cholerae variantstrain is used as a recipient strain for CTX phage infection.
 3. TheVibrio cholerae variant strain according to claim 1, wherein the Vibriocholerae variant strain uses a classical biotype, E1 Tor biotype,atypical E1 Tor biotype or O139 serotype Vibrio cholerae strain as aparental strain to prepare the Vibrio cholerae variant strain.
 4. TheVibrio cholerae variant strain according to claim 1, wherein the Vibriocholerae variant strain is a toxigenic Vibrio cholerae variant straincomprising one or more CTX prophages selected from the group consistingof CTX-1, CTX-cla, CTX-2, CTX-env, and CTX-O139 having a cholera toxingene.
 5. The Vibrio cholerae variant strain according to claim 1,wherein the Vibrio cholerae variant strain is a toxigenic Vibriocholerae variant strain comprising a cholera toxin gene inserted on thechromosome of the strain.